Plasminogen activators are a unique class of enzymes that convert the catalytically inactive zymogen plasminogen to its active enzymatic form, plasmin. Plasmin is a Serine Protease requisite for the dissolution of fibrin (Colleen, D., 1980, Thromb. Haemostasis 43:77-89). Several plasminogen activators are now being used as in vivo fibrinolytic agents in the treatment of acute myocardial infarction (Tiefenbrunn, et al., 1989, Fibrinolysis 3:1-15). Tissue plasminogen activator (t-PA) has been the focus of considerable attention because its ability to activate plasminogen is significantly enhanced in the presence of fibrin (Rijken et al., 1982, J. Biol. Chem. 257:1920-2925), a property that should make it more clot specific when used as an in vivo fibrinolytic agent.
Based on the cDNA sequence of t-PA, the inferred sequence of 527 amino acids comprises five distinct structural domains: a Finger domain; a Growth Factor domain; two Kringle domains; and a Serine Protease domain (Pennica et al., 1983, Nature 301:214, and Banyai et al., 1983, FEBS Lett. 163:37-41). A series of deletion mutants of t-PA that comprise the Serine Protease domain and one or more of the other four structural domains of the intact molecule have been constructed (van Zonneveld et al., 1986, Proc. Natl. Acad. Sci. 83:4670; Erlich et al., 1987, Fibrinolysis 1:75-81; Haigwood et al., 1989, Protein Engineering 2:611-620; Higgins and Bennett, 1990, Annu. Rev. Pharmacol. Toxicol. 30:91). A t-PA derivative lacking the Finger, Growth Factor and Kringle 1 domains has been described by Burck et al., 1990, J. Biol. Chem. 265(9):5170-5177). This derivative, called mt-PA6, was constructed by deletion of the DNA encoding amino acids 4-175 of human t-PA (t-PA). mt PA6 produced by recombinant DNA techniques was found to possess greater fibrinolytic activity and a greater ability to activate thrombus-bound plasminogen than t-PA (Jackson et al., 1990, Circulation 82:930-940).
mt-PA6 is found in two glycosylation forms when produced by eukaryotic cell culture using recombinant DNA techniques. These two glycosylation forms account for the doublet of 40 and 42 kD bands seen when the purified mt-PA6 molecule is analyzed by gel electrophoresis. One of the glycosylation forms, Primary mt-PA6 (mt-PA6-P), is glycosylated at amino acid 276 (equivalent to amino acid 448 of t-PA) but not at amino acid 12 (equivalent to amino acid 184 of t-PA). The second glycosylation form, Variant mt-PA6 (mt-PA6-V), is glycosylated at both amino acids 12 and 276. mt-PA6 lacks the Kringle 1 domain and, therefore, lacks the glycosylation site at amino acid 117 of this domain. mt-PA6-V comprises 10-25% of the mt-PA6 molecules secreted from the Syrian hamster cell line AV12-664 (Burck et al., supra).
The thrombolytic potential of mt-PA6-V and its role in fibrinogen degradation and plasminogen degradation were examined using methods described by Jackson et al., supra. These studies demonstrated that diglycosylated t-PA derivatives lacking the Finger, Growth Factor and Kringle 1 domains, such as mt PA6-V, provide advantages over their partially glycosylated counterparts in the treatment of thromboembolic disorders. Although the monoglycosylated mt-PA6-P has a higher in vitro specific activity, the diglycosylated mt-PA6-V is a more efficient thrombolytic agent in vivo. mt-PA6-V displays the unexpected and advantageous properties of causing less systemic conversion of plasminogen to plasmin, is markedly less prone to metabolism to its two-chain form, and has a higher fibrinolytic versus fibrinogenolytic ratio than mt-PA6-P. Surprisingly, mt-PA6-V also provides a greater maintenance of coronary blood flow and a faster time to reperfusion. Methods of treating thromboembolic disorders by the use of the diglycosylated form of t-PA derivatives that lack the Finger, Growth Factor and Kringle 1 domains, are disclosed by U.S. patent application Ser. No. 07/633,584, filed Dec. 24, 1990.
The present invention describes how these advantageous properties can be imparted to t-PA derivatives, such as mt-PA6, by means other than glycosylation. This is of importance because when these derivatives are produced by recombinant DNA technology from eukaryotic cell cultures, only 10%-25% of the resulting molecules are diglycosylated. The present invention makes it possible to convert the entire population of recombinantly produced t-PA derivatives that lack the Finger, Growth Factor and Kringle 1 domains to molecules having the enhanced thrombolytic properties of the diglycosylated form of the t-PA derivative molecule. Additionally, the economically disadvantageous process of separating the glycosylation forms in order to obtain only the diglycosylated form for use in in vivo thrombolytic therapy is avoided.
For the purposes of the present invention, as disclosed and claimed herein, the following terms are defined below.
Adduct--a combination of two or more independently stable compounds.
Amphipathic molecule--a molecule that has both polar and nonpolar portions, said nonpolar portion comprising a C.sub.9 --C.sub.15 straight or branched chain alkyl group and said polar portion comprising an anionic group.
Glycoforms--forms of a glycoprotein with unique biological activities resulting from differences in the composition of the N-linked glycosylation of the protein.
Glycosylation--the attachment of oligosaccharides to a protein through an N-glycosidic bond with the Asparagine residue in an Asn-X-Ser/Thr sequence, wherein X is any amino acid except proline.
Reperfusion--restoration of blood flow caused by successful thrombolysis.